NO753291L - - Google Patents

Abstract

Procedure for the preparation of new antibiotics.

Description

The present invention relates to a method for the production of new antibiotics, which is hereinafter referred to as SL 7810/F,

SL 7810/F-II and SL 7810/F-III, as well as their tetrahydro derivatives.

The peculiarity of the method according to the invention for the production of the new antibiotics and their tetrahydroderivatives,

is that

a) a strain of the genus Aspergillus rugulosus Thorn & Raper is grown in the presence of a nutrient medium, or b) for the production of the tetrahydroderivatives of these antibiotics, they are subjected to hydrogenation.

The new antibiotics and their tetrahydroderivatives have the following characteristics: SL 7810/ F

Colorless amorphous powder with melting point 160 - 163°C (decomposition);

/"o7p° 48.3°' (c = 0.806 in;; methanol).

Analysis: Found C 57.3 H 7.9 N 9.2 0 25.2%

Molar weight (determined osmometrically in ethanol): 1190

UV spectrum in methanol: Amax= 194 nm, log £' = 1.98

(see Fig. 1) 276 nm, log = 0.14

IR spectrum in nujol (see fig. 2)

<->1H-NMR in DMSO (100 MHz, tetramethylsilane as internal standard)

/"see fig. 3/.

The hydrolysis of SL 7810/F with 6 N hydrochloric acid (4 hours at 110°C under a N 2 atmosphere) gives, among other things, linoleic acid as a hydrolysis product.

(Solubility, stability, thin-phase chromatogram and MIC values see below).

SL 7810/F-II

Colorless amorphous powder with melting point 158 - 160°C (decomposition);

/α7p° =^7.9° (c = 0.81 in methanol).

Analysis: Found C 58.5 H 8.0 N 9.3 0 23.8%

Molar weight (determined osmometrically in methanol): 1018

UV spectrum in methanol: Å-max 193 nm, log = 1.95

(see Fig. 4) 278 nm, log^<*>0.18

TR spectrum in nujol (see fig. 5).

<\>H-NMR spectrum in DMSO (100 MHz, tetramethylsilane as internal standard) / see fig. 6/.

(Solubility, stability, thin layer chromatogram and MIC values see below.)

SL 7810/F-IIX

Colorless amorphous powder with melting point 164 - 168°C (decomposition);

/α7p° b -53.0° (c = 1.00 in methanol).

Analysis: Found C 61.1 H 8.0 N 9.0 0 22.0%

UV spectrum in methanol: /^max 193 nm, log £' = 1.98 (see Fig. 7) 278 nm, log = 0.24

IR spectrum in nujol (see fig. 8).

H-NMR spectrum in DMSO (100 MH&, tetramethyl silane as internal standard) /see fig. 9/.

(Solubility, stability, thin layer chromatogram and MIC values see below).

Tetrahydro- SL 7810/ F

Colorless amorphous powder with melting point 173 - 178°C (decomposition); after crystallization from ethanol-water (95 : 5) melting point 190 - 205°C / 210 - 212°C (decomposition);

Δ7d° = -47>8° (c = 0.984 in methanol).

Analysis: Found C 56.8 H 8.0 N 9.0 0 25.5%

UV spectrum in methanol: λ ma)<;s 194 nm, log £' = 1.85

(see Fig. 10) 277 nm, log =0.09

227 (shoulder)

log = 1.00

IR spectrum in nujol (see fig. 11)

H-NMR spectrum in DMSO (100 MHz, tetramethylsilah as internal standard) £see fig. 127.

<13>C-NMR spectrum in CD^OD with tetramethylsil an. as internal standard (see table 1)

(Solubility, stability, thin layer chromatogram and MIC values see below).

The hydrolysis of tetrahydro-SL 7810/F with 6 N hydrochloric acid (4 hours at 110°C under a N 2 atmosphere) gives, among other things, stearic acid as a hydrolysis product.

Tetrahydro- SL 7810/ F- II

Colorless amorphous powder with melting point 160 - 163°C (decomposition); /o7d° = -41.4° (c « 0.823 in methanol).

Analysis: Found C 58.7 H 8.1 N 9.6 0 23.7%

UV spectrum in methanol ^max <1>94 nm, log £, ' = 1.90

(see Fig. 13) 278 nm, log £, ' = 0.25

227 (shoulder)

log £' = 1.07

IR spectrum in nujol (see fig. 14)

\H^NMR spectrum in DMSO (90 MHz, tetramethylsilan - as internal standard) / see fig. 15.7.

13

C-NMR spectrum in CD^OD with tetramethylsilane as internal standard (see table 2).

(Solubility, stability, thin layer chromatogram and MIC values see below).

Tetrahydro- SL 7810/ F- III Colorless amorphous powder with melting point 175 - 180°C (decomposition); /bc7p°= -41.5° (c = 1.094 in methanol).

Analysis: Found C 60.5 H8.5 N9.2 0 21.9%-

UV spectrum in methanol: A max 193 nm, log £11.93

(see Fig. 16) 278 nm, log = 0.19

224 nm, log 1.01 (shoulder)

IR spectrum in nujol (see fig. 17)..

H-NMR spectrum in DMSO (100 MHz, tetramethylsilane as internal standard) / see fig. 18J7.

(Solubility, stability, thin layer chromatogram and MIC values see below).

Solubility

The antibiotics SL 7810/F, SL 7810/F-II and SL 7810/F-III as well as their tetrahydroderivatives are at room temperature easily soluble in methanol and ethanol, poorly soluble in chloroform, ether, petroleum ether, hexane and practically insoluble in water.

Stability

The new antibiotics SL 78.10/F, SL 7810/F-II and SL 7810/F-III are unstable in the presence of light and especially in air and decompose. The splitting is also noticeable when heated above 50°C. The hydrogenation to the tetrahydro derivatives largely eliminates the instability.

Characteristic of all three antibiotics and their tetrahydroderivatives is their instability under basic conditions. At a pH above 8, conversion to cleavage products occurs which are biologically only weakly active or are no longer active.

Thin layer chromatogram

Analysis takes place on silica gel mark 60 plates (0.25 mm layer thickness). With the following eluents, the following R^ values can be determined:

The antibiotics SL 7810/F, SL 7810/F-II and SL 7810/F-III show in this determination the same R^ values as their tetrahydro derivatives.

When developing with a 0.2% Ce(SO^)2~ solution in 50% sulfuric acid as a shower reagent, the following spots (after heating at, 130°C) can be detected:

Iodine vapor can also be used for detection of the antibiotics SL 7810/F, SL 7810/F-II and SL 7810/F-III.

The separation between the three antibiotics SL 7810/F, SL 7810/F-II and SL 7810/F-III from their tetrahydroderivatives can also be done by thin-layer chromatography on silica gel -GF 254 plates (0.3 mm layer thickness), as the silica gel is mixed with 3% AgNO^. With chloroform-methanol-water (70 : 25 : 5) as eluent, the following R^-values can be determined with 40ifsubstance:

To detect the substances, shower with vanillin-sulphuric acid.

When heated to 120 - 130°C, yellow-brown color spots appear for the tetrahydro compounds. The non-hydrogenated compounds show a reddish colouration.

Method a) can be carried out in known ways for fermentative production of antibiotics. A culture thereof was deposited in the United States Department of Agriculture (Northern Utilization Research and Development Division), Peoria, 111., USA and is available to the public under the designation NRRL 8039. The strain NRRL 8039 is preferably used.

However, strains can also be used which have been extracted from the starting strain aspergillus rugulosus Thorn & Raper by treatment with mutagenic substances or radiation or by selection.

Characteristics of strain NRRL 8039

The new strain NRRL 8039 of aspergillus rugulosus Thorn & Raper was isolated from a soil sample isolated on the Jaum Pass in Switzerland and, due to its morphological features, allows the aspergillus monograph by K.B. Raper & D.I. Fennell (THE GENUS ASPERGILLUS: The Williams & Wilkings Company Baltimore, 1965, page 686) in

.under Aspergillus rugulosus Thom_':& Raper.

The strain NRRL 8039 grows on Agar nutrient substrate between 15 and 50°C, the growth optimum being between 35 and 42°C. On Czapek-Dox-Agar, the strain NRRL 8039 grows rather slowly, the cultures usually correspond approximately to the species description (Raper & Fennell, 1965). : 20-day-old colonies are strongly wrinkled and the grey, velvety aerial mycelium is grey, pigmented light brown to brownish violet. At lower incubation temperatures (e.g. 18 - 27°C), the edge of the colony becomes white, while at higher temperatures (e.g. 33 - 45°C) it appears yellowish brown. The backside of the colony becomes yellowish at lower incubation temperatures, at higher temperatures a violet pigment is formed and secreted into the agar. Also after 20-day incubation on Czapek-Dox-Agar no Cleistothecia are formed and only at higher incubation temperatures simple but stunted conidia carriers.

Significantly faster and better developed, the strain NRRL 8039 on 2% malt-dextrose agar, where already after 20 days of incubation at 27°C the main and secondary fruit forms are formed in considerable quantities and mature. The colonies are at most quite weak, but most often at all: not wrinkled, and the velvety aerial mycelium is uniformly yellowish in colour. The back of the colony is yellow at lower incubation temperatures and light brown at higher temperatures.

The strain NRRL 8039 growing on 2% mali-dextrose agar shows small dark green conidial heads, which most often appear in smaller groups in and on the edge of the colony.

The morphology and dimensions of all structures of the bee fruit forms agree well with the aforementioned species description of aspergillus rugulosus. With regard to the main fruit shape, however, there are some differences in relation to the species description: thus the globular Cleistothecia that develop in the Slere layer of the velvety aerial mycelium are in large numbers significantly smaller than what is indicated for the species (50.- 80 ju in diameter in compared to 225 - 350 /u) . The enveloping cells are also vitreous, whereas in this species they should be dark brown. A good agreement with the species description, on the other hand, consists in the characteristic feature of them. purple-orange-red, lenticular 4.1 - 5.0 x 3.4 - 3.8 p large ascos<p>ores, whose surface is more or less distinctly wrinkled and furrowed and whose two parallel equatorial sums are 0.4 - 0 ,7|U wide. ).

The new strain NRRL 8039 can be grown on different nutrient substrates with the usual nutrients, e.g. as described in the following 'example.

The new antibiotics SL 7810/F, SL 7810/F-II and SL 7810/F-III can be prepared in such a way that a liquid medium is inoculated with a spore or mycelial suspension of iammen NRRL 8039 and the culture is incubated for 2 - 4 days, preferably 3 days, at 27°C. Cultivation takes place under aerobic conditions, either in surface or submersible conditions. As soon as <-'a maximum quantity of antibiotics has been produced (can be ascertained by the detection of the activity against Candida albicans) these are isolated from the culture broth according to methods known per se, e.g. as described in the following example, where other organic solvents are also used as the solvent substitute for ethyl acetate, such as e.g. butyl acetate or butanol.

For separation from fatty secondary substances from the raw extract, a filtration over a silica gel column can be carried out as a first operation, with approximately the 4-5 times the amount of silica gel by weight, calculated on the amount of extract, being used. The elution takes place with chloroform-methanol mixtures (see the following example).

Another method can e.g. carried out in the following way: Fatty by-products and other by-products are separated as appropriate ' fel1ing reactions. For this, the ethyl acetate extract is rubbed with petroleum ether, as the antibiotics active against candida/albicans remain in the insoluble part. This residue is slurried in imethanol and, after filtering off insoluble inactive parts, the filtrate is evaporated in a vacuum. This material is dissolved in methanol and the clear solution is added dropwise to the 10-fold amount of ethyl acetate. The precipitate contains SL 7810/F, SL 7810/F-II and SL 7810/F-III alongside inactive by-products. The further purification and separation of the components from the mixture now takes place using chromatographic methods known per se, whereby gel chromatography on . "Séphadex LH 20" and chromatography on silica gel are very suitable. These methods are used in combination and for cleaning the individual components. Thus, e.g. chromatographed one of the above-described raw products first on "Séphadex LH 20" with methanol, the three antibiotics appearing in the elution in the same fractions.

The combined active fractions are then chromatographed on silica gel, grain size 0.06 - 0.2 mm. Either chloroform can be used for elution, to which increasing amounts of methanol are added, but also a chloroform-methanol-water mixture (80 : 17.5 : 2) can be used throughout. The corresponding R^ values in the thin-layer chromatogram are obtained

in the elution from the silica gel columns first SL 7810/F-III,

then SL 7810/F-II and finally SL 7810/F. For separation of mixture fractions, especially SL 7810/F-II and SL 7810/F-III, chromatograph if necessary several times on the 250- to 400-.in double amount of silica gel. The fractions active against Candida albicans and pure in thin-layer chromatograms correspond to the R^ values for the antibiotics in question.

Procedure b) can also be carried out in and of itself

known methods, e.g. e.g. \ved_ Catalytic hydrogenation .

Preferred solvents are lower aliphatic alcohols, such as e.g. methanol or ler.</> ethanol, in speech. The hydrogenation takes place, suitably in a neutral or weakly acidic area, e.g. in the presence of weak'organic acids such as acetic acid. The hydrogenation takes place at ■ temperatures between 10 and 40°C, preferably between 20 and 25°C. The hydrogenation conveniently takes place under atmospheric pressure or slightly elevated pressure, i.e. in the presence of a platinum catalyst, such as for example PtC^, which is prehydrogenated to Pt, or preferably a palladium catalyst, such as for example palladium on charcoal or barium sulphate. The hydrogenation takes place until complete hydrogen uptake. For this, it is assumed that the hydrogen uptake corresponds to 2 mol per mol of antibiotic SL 7810/F, SL 7810/F-II and SL 7810/F-III with a molecular weight of approx. 1000 to 1200. In the case of hydrogenated derivatives of the mentioned antibiotics, no protons indicating linoleic acid could be detected in the NMR spectrum.

They obtained hydrogenated derivatives of the antibiotics SL 7810/F,

SL 7810/F-II and SL 7810/F-III are cleaned if necessary in a manner known per se. Chromatography on "Séphadex LH 20" (elution with methanol) and/or chromatography on fine-grained silica gel is preferably suitable for this, as it is suitably eluted with chloroform-methanol mixtures (9:1) to (3:1) or with chloroform-methanol mixtures while adding of a small amount of water.

The present invention also includes fermentation solutions, which are obtained by cultivating a strain of the genus aspergillus rugulosus Thorn & Raper which produces one of the antibiotics-; SL 7810/F, SL 7810/F II and/or SL 7810/F-III.

The new antibiotics and their tetrahydroderivatives show interesting pharmacological properties.

For biological characterization of the antibiotics > SL 7810/F, SL 7810/F-II, SL 7810/F-III and their tetrahydroderivatives serve their spectrum of action. While all compounds under the usual test conditions are inactive against gram-positive and gram-negative bacteria such as e.g. staphylococcus aureus and escherichia coli, show distinct effects against yeast and fungal species.

In the following table are the minimum inhibitory concentrations

against various yeast and fungal species listed. The serial dilution test used for this was carried out in a known manner with the indicated test strains. Incubation in the serial dilution test took place in a malt extract medium (2%) with pH 4.4 - 4.7 at 27 to 37°C depending on the temperature optimum for the test strain in question. The assessment took place after 72 hours of incubation. The test strains

is deposited in the mycotheque of the company Sandoz and is available there.

As can be seen from the above results (MIC values) show

the aforementioned compounds an activity against specific fungal species,

especially against candida albicans. The effect against candida albicans can also be determined in vivo by the following tests:

1. Experimental intestinal infection with candida albicans

A 124 in female mice

.--.With this method, the equilibrium in the normal intestinal flora is disrupted to the advantage of orally administered candida albicans^124. With the broad-spectrum antibiotic treatment, suppression of the bacterial flora occurs and thus to the selective reproduction of candida-s-.opp. scars in the intestine of the female mice. The Candida organisms cultivated in the intestine are excreted in the faeces (, bacterial count 10 4 /g faeces). regularly during at least three weeks.

Material and method

Laboratory animals:

Female NMRI mice are used which were fed ad libitum with mouse pellets and which had a weight of 18 - 24 g. In the drinking water it was added

1 mg/ml streptomycin.

5l22!syl££i!22_22_in^'e'<:s ion •

The intestinal infection of the female mice with candida albicans was carried out

in the following manner:

From a vial stored in liquid nitrogen and thawed, a dilution containing 5 x 10 7 germs in 0.25 ml was prepared.

It became so ° in it. in some cases added .5 x 10 7 germs per mice orally After the infection, the animals were distributed individually in cages (6 or 10

animals kept individually constitute a collective).

!5i!I!;!:^Iikestemmelmes_in excrement n9 (KZ/g excrement) :

The experimental animals were on the 6th day - respectively at the time when the germ count in the faeces is to be determined, placed in sterile macrolon cages and faeces for 4 hours were collected and weighed. A suspension of 10 - 1 was then prepared in a NaCl solution and diluted to 10 -7. The germs were then counted in the plate method with Saboraud agar, as per ml of SAB agar was added with 50 µg of streptomycin. When streptomycin was added to the agar, the growth of the other intestinal germs was suppressed and a pure culture of the fungus Candida albicans was obtained on the agar plate.

Dosage_and method of treatment:

The highest administered dose of the test substance generally follows the dose tolerata maxima. It amounts to a maximum of 300 mg/kg body weight for mice. A further dosage amounts to 100 mg/kg body weight.

These daily supply doses are administered orally for 5 days, beginning on the day of the infection. The daily doses are divided into three single doses and administered orally in a volume of 0.25 ml (for water-insoluble substances, 0.2% KMC + 0.2% "Tween 80" serve as suspension agents) to 20 g mice.

Each infection trial was completed by including 10 control animals, which received a standard substance. As a standard substance, "Nystatin" was added in daily doses of 300 and 100 mg/kg body weight orally in this infection experiment. The supply of these doses took place as with the test substance. At a p.o. given a dose of 300 mg/kg body weight "Nystatin", one day after the end of treatment it is not possible to detect any germs of Candida albicans in the faeces, while 100 mg/kg body weight "Nystatin" causes a germ reduction of at least two tier powers, in some cases also already a complete elimination of these germs.

For each dosage there is a group of 6 animals each, which are kept individually in cages. 6 additional animals, like all others, also receive streptomycin in the drinking water and the solvent without substance as a control. The animals are also kept individually.

Determination and assessment:

When determining such an experiment, reductions in germ counts were respectively germ eliminations determined and compared with the untreated control animals or compared' with the standard substances' control animals

and calculated as a percentage.

2. Vaginal Candida albicans infection in rats

(stem Al24)

Vaginal mycosis in female rats is a mucosal infection. An experimental vaginal mycosis can be produced by ovariectomy and subsequent hormone administration to induce permanent estrus in the sexually mature rat. This local infection covers approximately only the outermost surface layers of the vaginal mucosa. With smears, its progress can be carefully controlled. The good reproducibility and the uniform course of the infections meet all the requirements that are the prerequisite for the search for a new active ingredient.

Laboratory animals^

Wistar rats with a weight of approx. 200 g. The animals received food and water ad libitum. For each experimental group,

10 animals were used.

Preparation and implementation of the infection:

Under "N.embutal" anesthesia, the animals were ovariectomized. IN'; One day after the ovariectomy, 100 µg estradiol was added subcutaneously in a quantity of 0.5 ml sesame oil. rat. One week after the hormone addition, the animals were in an estrus state, as this could easily be demonstrated using smears and methylene blue staining under a microscope.

In this state of varigc:browns t there can hardly be leucocytes in the vaginal contents, as this is an advantage for the manifestation of an infection, as germs that penetrate the vagina are not phagocytosed by the leucocytes. 7 days after the ovariectomy, the infection occurs. With a tuberculin syringe and throat probe, Candida albicans germs are added in a quantity of 0.05 ml or 10^ germs per rat intravaginally.

The production of inoculum takes place according to usual methods and is stored

1 liquid nitrogen. The germs are used directly from after rapid thawing for the infection. The course of the local vaginal mycosis is checked on the 3rd, 7th, 10th, 15th and 20th days after the infection. Vaginal contents are smeared with a platinum ring on str eptomycin-containing Sabouraud-agar plate (20 µg/ml). In parallel, smears are prepared with vaginal contents which are stained for Gram detection and examined under the microscope for the presence of pseudomycelium. After 2 days of cultivation of SAB plates (tubes) at 30°C, these are assessed semi-quantitatively. Depending on the growth of candida albicans colonies, one can speak of a high-grade (rapid growth), moderate (countable colonies) and low-grade (single-growth of colonies of candida albicans) infection .

Handling_and_dosing_:

The local treatment of the experimental vaginal caddisiasis begins 24 hours after the infection. Intravaginal therapy is given twice a day (morning and afternoon) for 5 consecutive days.

As a single dose of a test substance is administered in the individual case

0.5 ml of a 1.5% and a 5% ointment per rat.

The substances are processed in a mixture of 2 parts • polyethylene glycol

300 and 1 part polyethylene glycol 1500 "ointment base" and administered intravaginally.

Each infection trial was supplemented by the inclusion of a group

on 10 animals as control animals, which received a known standard substance. As a standard substance, "Nystatin" was used as a 1.5% ointment or alternatively "Clotrimazole" as 1.5% ointment. 10 additional animals received ointment base without substance and a group of 10 animals serves as infection control1.

The new antibiotics and their tetrahydroderivatives are therefore suitable for the treatment of diseases caused by Candida albicans.

Satisfactory results are usually obtained with a daily dose of approximately 100 to 1500 mg. If necessary, this dose can be administered i

2 to 3 servings of 30 to 750 mg.

As medicine, the new antibiotics and their tetrahydroderivatives can be administered alone or in suitable preparation form together with inorganic or organic, pharmacologically indifferent excipients. E.g. they are used as a component of an ointment, as the concentration in the ointment can amount to approx. 5 to 130 mg of active ingredient per gram of ointment. Other forms of preparation are tablets; capsules and solutions.

In the following examples to illustrate the invention,

All temperature indications are in degrees Celsius. All stated ratios are on a volume basis.

Example 1:

A spore suspension that serves for inoculation of the nutrient solution is produced from a culture of strain NRRL 8039. For this, 200 ml of nutrient agar with the following composition is inoculated:

After 10 days of incubation at 2.7°C, the formed spores are suspended

in 200 ml of sterile water and a standing fermentation vessel containing 50 liters of the following nutrient solution, inoculate the following:

B as well as 10% polypropylene glycol 2020 (Chem. Werke Huls, Mari

BRD)

1 liter of de-ionized water.

This fermentation container was incubated with stirring for 42 hours at 27°C, with the aeration amounting to 1 liter of air/min/liter of medium, at a stirring speed of 250 rpm.

This culture serves as a pre-culture for 500 litres.^the fermentation vessel -with

the same nutrient solution which was thus incubated for 66 hours at 27°C. The aeration was again 1 liter of air/min./liter of medium, but the stirring speed was only 150 rpm. After 66 hours of incubation, the culture broth was harvested and worked up as follows: 450 liters of culture broth were adjusted to pH 7.0 with 2 N hydrochloric acid, 500 liters of ethyl acetate were added and homogenized with a "Dispax" reactor. The organic phase was then separated from the broth using a separator and washed with 50 liters of water. This extraction step

was repeated twice. The 3 extracts obtained were combined and evaporated to dryness under vacuum (water ring pump) at 20 to 40°C.

This material is fractionated on the 4-fold amount of silica gel "Mark 60" (grain size 0.063 - 0.20 mm). The eluates with chloroform + 10% methanol and chloroform + 20% methanol contained inactive secondary substances. The chromatography column was then eluted with chloroform-methanol (1:1) and the fractions with activity against Candida albicans were combined and evaporated to dryness. By chromatographing 16.4 g of active material on 1 kg of silica gel "Mark 60" (grain size see 0.063 - 0.20 mm) and elution with chloroform + 20% methanol, mixed fractions of SL 7810/F-II were first obtained and SL 7810/F-III. The subsequent elution with chloroform + 30% methanol gave fractions

-with SL 7810/F as main component. After gel filtration of SL 7810/F fractions (8.1 g) on 1.2 kg "Séphadex LH 20" with methanol, SL 7810/F was recovered in almost pure form. This preparation was dissolved in acetone while heating and the clear solution was evaporated to 1/4 of the volume. After standing for 3 hours at -15°G, the formed is filtered off

precipitate and dried after washing with acetone and ether for 24 hours at room temperature above & 2®5 ^ high vacuum. SL 7810/F is obtained as a colorless powder with a melting point of 160 - 163°C (decomposition).

By continued chromatography of the active mixture fractions of SL 7810/F-II and SL 7810/F-III from the above-mentioned silica gel chromatography, the two atatibiotics could be isolated. For this, the material (2.5 g) is dissolved in chloroform + 20% methanol and the solution was absorbed on a column with 1 kg of silica gel, which was treated with the same solvent mixture. The elution took place throughout with chloroform + 20% methanol, with first predominantly SL 7810/F-III being eluted and then predominantly SL 7810/F-II. For the extraction of pure SL 7810/F-III, the evaporated mixed fractions (0.75 g) containing this antibiotic are dissolved in chloroform + 10% methanol and the solution is adsorbed analogously to what was previously described on 1 kg of silica gel "Mark". The elution with chloroform + 10% methanol to 15% methanol gave the highly enriched material of SL 7810/F-III. After Éjelf il trituration (0.55 g) of 500 g "Séphadex LH 20" with methanol, pure SL 7810/F-III is obtained: Colorless amorphous powder with melting point 164 - 168°C (decomposition).

Before isolating SL 7810/F-II, the mixture fractions (1,7.£.§) containing SL 7810/F-II are dissolved in chloroform + 20% methanol and this solution is adsorbed on a column treated with the same solvent blanking with 1 kg silica gel. The elution with chloroform + 20% methanol gives strongly enriched fractions of SL 7810/F-II. The subsequent gel filtration (1.2 g) of 1.2 kg ^Séphadex LH 20" with methanol gives pure SL 7810/F-II, an amorphous powder with melting point '158 / 160°C (decomposition).

Example 2: Tetrahydro-SL 7810/ F

A suspension of 1 g of palladium charcoal (10% Pd) in 250 ml of alcohol or 250 ml of alcohol-glacial vinegar (1:1) is prehydrogenated for 30 min. A solution of 5 g of SL 7810/F in 50 ml of alcohol-glacial vinegar is then added

(1 : 1). After 3 hours of hydrogenation at 20°C under atmospheric pressure, the catalyst is separated by filtration and the clear filtrate is evaporated in a vacuum. The evaporation residue is dissolved in chloroform-methanol

(l : 1), with this solution impregnate 10 g of silica gel 60 "Mark"

(grain size 0.063 - 0.20 mm) and this is introduced onto a column with 500 g of silica gel. The subsequent elution with chloroform-methanol (4:1) gives the amorphous hydrogenation product as a colorless powder with a melting point of 173 - 178°C (decomposition). The preparation is crystallized from ethanol-water (95:5). Melting point after drying for 3 hours in high vacuum at 50°C: 190 - 205°C / 210 - 212°C (under decomposition).

Example 3: Tetrahydro-SL 7810/F-II

, The preparation takes place analogously to example 2, in that antibiotic SL 7810/F-II is used as starting material.

Example 4: Tetrahydro-SL 7810/F-III

The preparation takes place analogously to example 1 2, in that the antibiotic SL 7810/F-III is used as the starting material, the hydrogenation duration was 4 hours and as the solvent mixture for the chromatography on silica gel chloroform-methanol-water (80 : 17.5 : 2).

Claims (4)

1. Process for the production of the new antibiotics SL 7810/F, SL 7810/F-II, SL 7810/F-III and their tetrahydroderivatives, characterized in that a) a strain of the genus Aspergillus rugulosus Thorn & Raper is cultivated in the presence of a nutrient medium, or b) one of the antibiotics SL 7810/F, SL 7810/F-II and SL 7810/F-III is hydrogenated. 2. Method as stated in claim 1,'' characterized in that a strain is used which is extracted from the starting strain Aspergillug. rugulosus Thorn & Raper by treatment with mutagenic substances or radiation or by selection. 3. Method as stated in claim 1, characterized in that the strain NRRL 8039 is used as a new strain of the genus aspergillus rugulosus Thorn & Raper. 4. Fermentation solutions obtained by cultivation of a strain of the genus aspergillus rugulosus Thorn & Raper which produces antibiotics SLS7810/F and/or SL 7810/F-II and/or SL 7810/F-III.